Lepidopteran insect resistant transgenic potato plants

ABSTRACT

Lepidopteran insect resistant transformed potato plants are described. In particular, the transformation of potato plants with five to ten copies of a translational fusion encoding the 68 kD lepidopteran-specific toxin from Bacillus thuringiensis var. kurstaki HD-73 and neomycin phosphotransferase II is described. The transgenic potato plants were resistant to the tobacco hornworm, a lepidopteran insect which is susceptible to Bacillus thuringiensis var. kurstaki toxin.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

The present invention relates to transgenic potato plants (Solanumtuberosum) which are lepidopteran insect resistant. In particular, thepresent invention relates to potato plants containing multiple repeatingsegments of DNA which encode an endotoxin from Bacillus thuringiensis sothat the potato plant is provided with the insect resistance.

(2) Prior Art

It is well known that the bacterium Bacillus thuringiensis (B.t.)produces proteinaceous parasporal inclusions during sporulation. TheseBacillus thuringiensis proteins are toxic and highly specific to certainlepidopteran, coleopteran, and dipteran insects (Hofte, H. and H. R.Whiteley, Microbiological Reviews, 53, 242-255 (1989)). Crystal andspore preparations from B. thuringiensis var. kurstaki (B.t.k.) havebeen used for many years as insecticides to control a variety oflepidopteran insects (Dulmage, H. T., Microbial Control of Pest andPlant Diseases 1970-1980 in: H. D. Burges (ed), Academic, London, pp.193-222 (1980)). The protoxin from Bacillus thuringiensis var. kurstakihas a size of 130 kD, which is converted to a biologically activepolypeptide of 68 kD in the midgut of susceptible insects (Bulla, L. A.,Jr., et al., Biochem. Bio Phys. Res. Commun. 91, 1123-1130 (1979); andAdang, M. J., et al., Gene, 36, 289-300 (1985)).

Because Bacillus thuringiensis proteins have short half-lives whenapplied topically (Beegle, C. C., et al., Environ. Entomol., 10, 400-401(1981)), introduction of Bacillus thuringiensis genes into plants couldbe a better utilization of this biological pest control system.Transgenic tobacco, tomato, and cotton plants containing Bacillusthuringiensis var. kurstaki genes have been reported to be resistant toattacks from target lepidopteran insects (Perlak, F. J., et al.,Bio/technology 8, 939-943 (1990); Barton, K. A., et al., Plant Physiol.85, 1103-1109 (1987); Vaeck, M., et al., Nature, 328, 33-37 (1987); andDelannay, X., et al., Bio/Technology, 7, 1265-1269 (1989)). Hornworm hasbeen found to be susceptible to the Bacillus thuringiensis var. kurstakiencoded toxin in tobacco (MacIntosh, S. C., et al., J. InvertebratePathology, 56, 258-266 (1990)).

The problem is to provide the DNA encoding the protein in potatoes suchthat a sufficient amount is encoded in the plant to make it resistant tothe lepidopteran insects. This has not been accomplished by the priorart.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide noveltransgenic potato plant which are resistant to Lepidopteran insects. Itis further an object of the present invention to provide the transgenicplants which contain multiple repeating tandem segments of Bacillusthuringiensis DNA encoding an endotoxin or endotoxins. These and otherobjects will become increasingly apparent by reference to the followingdescription and the drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram of the plasmid pWB139 where RB is right border ofthe T-DNA; Nos is the nopaline synthase promoter; CaMV 35S is thecauliflower mosaic virus 35S promoter; btk is the DNA sequences encodingthe 68 kD B.t.k. toxin (only codons 1-612 from the full length gene wereused); nptII is the DNA sequences encoding neomycin phosphotransferaseII (in a frame translational fusion following the btk codons); tom PI 3'is the tomato proteinase inhibitor I transcription terminator; nos 3' isthe nopaline synthase transcription terminator; * is the unique site inpWB139; WB241 and WB242 are primers used in PCR-amplified DNA and MB23and MB24 are primers used in PCR-amplified cDNA. The distance betweenWB241 and WB242 is 1090 bp and between MB23 and MB24 is 720 bp. The leftborder of the T-DNA has been removed. The abbreviation "bp" is basepairs.

FIGS. 1A-1 to 1A-3 show the DNA sequence of the B.t.k. gene in pWB139encoding the truncated endotoxin.

FIG. 2 is an autoradiogram of Southern blot of PCR amplified DNA frompWB139, untransformed FL1607, and seven regenerated potato plantscapable of rooting in medium containing 50 mg/l kanamycin. PCR wasperformed using a pair of primers (WB241 and WB242) amplifying a 1090 bpregion of the translational fusion. After PCR, the samples weresubjected to electrophoresis in a 1% (w/v) agarose gel at 5 v/cm for 1.5hours (DNA migration was from top down). Amplified DNA was blotted ontoa Nytran membrane, and hybridized to a ³² P-labeled 2.6 kb Hind IIIfragment of pWB139 covering the entire region of the translationalfusion. BstE II digested lambda DNA was used as the size standard. Lane1: DNA homologous to the probe was amplified using pWB139 DNA as thetemplate; lane 2 to 4: DNA homologous to the probe was amplified usingtotal DNA extracted from three regenerated FL1607 plants as thetemplate; lane 5 to 8: DNA homologous to the probe was not amplifiedusing DNA extracted from four regenerated FL1607 plants; lane 9: DNAhomologous to the probe was not amplified using DNA from untransformedFL1607. bp=base pairs.

FIG. 3 is an autoradiogram of a Southern blot of pWB139, as well astotal DNA from untransformed and transformed FL1607 plants. 250 μg ofpotato DNA was digested with 150 units restriction enzyme at 37° C.overnight. The digests were subjected to electrophoresis, transferred toa Nytran membrane, and hybridized to the ³² P-labeled 2.6 Kb Hind IIIfragment of pWB139. DNA migration was from top down. BstE II-digestedlambda DNA was used as the size standard. 250, 1250, and 2500 pg ofpWB139 DNA corresponding to 1, 5, and 10 copies of the translationalfusion in 250 μg potato DNA were used to estimate the copy number of theintegrated btk:npt gene fusion. Lane 1: 2500 pg Bam HI-digested pWB139DNA; lane 2: 1250 pg Bam HI-digested pWB139 DNA; lane 3: 250 pg BamHI-digested pWB139 DNA; lane 4 and 5: Hind III-digested total DNA fromuntransformed and transformed FL1607, respectively; lane 6 and 7: BamHI-digested total DNA from untransformed and transformed FL1607,respectively. kb=kilobase pairs.

FIG. 4 is an autoradiogram of Southern blot of PCR-amplified cDNAhomologous to the translational fusion in transgenic FL1607 and tobaccoplants. cDNA was synthesized from purified poly A+ mRNA, thenPCR-amplified using a pair of primers (MB23 and MB24) which amplify a720 bp region of the translational fusion. Amplified DNA was subjectedto electrophoresis in a 1% (w/v) agarose gel, blotted onto a Nytranmembrane, and hybridized to a ³² P-labeled 2.6 kb Hind III fragment ofpWB139. DNA migration was from top down and BstE II digested lambda DNAwas used as the size standard. DNA contamination in mRNA preparationswas checked by parallel cDNA synthesis and PCR without reversetranscriptase. Lane 1: PCR-amplified DNA homologous to the probe usingpWB139 DNA as the template; Lane 2: DNA homologous to the probe was notamplified from transgenic tobacco when cDNA synthesis was performedwithout reverse transcriptase; lane 3: PCR-amplified DNA homologous tothe probe using cDNA from transgenic tobacco as the template; lane 4:DNA homologous to the probe was not amplified using cDNA fromuntransformed FL1607 as the template; lane 5: DNA homologous to theprobe was not amplified from transgenic FL1607 when cDNA synthesis wasperformed without reverse transcriptase; lane 6: PCR-amplified DNAhomologous to the probe using cDNA from transgenic FL1607 as thetemplate.

FIG. 5 is leaf area consumption by tobacco hornworm (Manduca sexta) in apetri dish (5 cm diameter) after 24 hours in laboratory tests. Left row:leaf disks (2 cm diameter) taken from an untransformed FL1607 plant;right row; leaf disks (2 cm diameter) taken from the transformed FL1607plant.

FIG. 6 is a photograph showing the growth of first instar larvae ofhookworm over 24 hours. The upper insect was fed on control leaf discand the lower two (2) insects were fed on the transgenic potato leafdiscs for 24 hours. The lower two (2) larvae are stunted.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a transformed potato plant containingDNA encoding a Bacillus thuringiensis endotoxin repeated multiple timessuch that the plant is resistant to Lepidopteran insects. Further thepresent invention relates to a method for transforming a potato plantwhich comprises: transforming cells of the potato plant with DNAencoding a Bacillus thuringiensis endotoxin which inhibits Lepidopteraninsects; and isolating the transformed plants which are resistant to theLepidopteran insects wherein the DNA is repeated multiple times in thetransformed cells. It was unexpected that the transformed plant would beviable and insect resistant.

The fact that Bacillus thuringiensis (B.t.) produces endotoxins whichare active against insects is well known. The endotoxins have beensequenced so that their chemical structure is known. Further, the DNAsequences which code for the endotoxins are well known. The mostpreferred DNA encoding the endotoxin is from Bacillus thuringiensis var.kurstaki (B.t.k.) or an active segment thereof.

The Bacillus thuringiensis DNA can contain marker DNA which allows forselection of the transformed plant. Antibiotic markers, particularlyneomycin phosphotransferase II, are preferred. The plasmid pWB139(FIG. 1) is preferred.

The DNA to be transformed is contained in a plant bacterium well knownfor its ability to infect plants and is referred to as a "vector". TheBacillus thuringiensis DNA and marker DNA are contained in Agrobacteriumtumefaciens as the vector. The DNA also contains promoter DNA whichpromotes the transcription of the Bacillus thuringiensis DNA.

The binary plasmid pWB139 contains, besides the translational fusionincluding the BtDNA, the regulatory regions that originate from theplasmid pWB146 (Barnes, W. M., Variable patterns of expression ofluciferase in transgenic tobacco leaves. Proc. Natl. Acad. Sci. US, v.87, 9183-9187 (1990)). They include the NOS gene promoter, the 35Spromoter from CaMV and the 3' regulatory regions from the tomatoproteinase inhibitor I gene (Lee, J. S., et al., Molecularcharacterization and phylogenetic studies of a wound-inducibleproteinase inhibitor I gene in Lycopersicon species. Proc. Natl. Acad.Sci. USA, v. 83, 7277-7281 (1986)) and from the NOS gene. Both 3'nontranslated regions contain the sequences typical for poly A signals(AATAAG in Tom PII and AATAAT in NOS). Although the function(s) of poly(A) is still not completely understood, evidence obtained mostly inmammalian and yeast systems suggests that polyadenylation may beinvolved in regulating several processes: nuclear processing andtransport, modulation of mRNA translational efficiencies and mRNAturnover (Atwater, J. A., et al., Regulated mRNA stability. Annu. Rev.Genet., v. 24, 519-541 (1990) and Brawermann, G., The role of poly (A)sequence in mammalian messenger RNA. CRC Crit. Rev. Biochem. v. 10, 1-38(1981)). For the Tom PII gene it was demonstrated that wound inducibleresponse of the gene is dependent upon the presence of the 3' regulatoryregion (Barnes, W. M., Variable patterns of expression of luciferase intransgenic tobacco leaves. Proc. Natl. Acad. Sci. US, v. 87, 9183-9187(1990)). When tested in transgenic tobacco plants the presence of thisregion correlates with the tissue-specific expression governed by theregulatory region (Barnes, W. M., Variable patterns of expression ofluciferase in transgenic tobacco leaves. Proc. Natl. Acad. Sci. US, v.87, 9183-9187 (1990)). The transgenic plant can be mated with otherpotato plants to transfer the foreign DNA to the progeny where it isexpressed. In this manner preferred potatoes having improvedcharacteristics can easily be produced.

The insect resistance of the plants is evidenced by the lowerconsumption by the insects of the plant leaves where the DNA encodingthe Bacillus thuringiensis endotoxin resides. It was unexpected that theBacillus thuringiensis DNA would be sufficiently expressed to affect theinsects. The reason for this result is that the DNA is encoded bymultiple repeating units of the DNA (between 5 and 10) in the potato,particularly the leaves.

EXAMPLES Example 1

Summary of Findings

Solanum tuberosum section FL1607 obtained from Frito Lay, Inc.,Rhinelander, Wis., was transformed using an Agrobacterium tumefaciensbinary vector containing DNA encoding a translational fusion between apart of the Bacillus thuringiensis var. kurstaki (B.t.k.) HD-73δ-endotoxin and neomycin phosphotransferase II (NPT II). The bt segmentfor HD-73 is between nucleotide 387 and nucleotide 2233 as described inAdang et al, Gene 36, 289-300 (1985). The segment includes the first 612codons of this bt gene. Codons 3, 4 and 5 are altered to provide aHindIII restriction site in the bt gene of pWB139. The gene sequence andencoded endotoxin are shown in FIGS. 1A-1 to 1A-3. Two hundred andforty-three shoots were regenerated from fifty inoculated leaf explants.Putatively transformed shoots were selected based on their ability toroot in a growth medium supplemented with 50 mg/l kanamycin. DNAsequences encoding NPT II were detected using the polymerase chainreaction in several plants which formed roots in the selection medium.Southern blot hybridization of total DNA isolated from agreenhouse-grown transformed plant demonstrated integration of thetranslational fusion into the plant genome. In addition, transcriptionof the gene fusion in the transgenic potato plant was detected byreverse transcription of mRNA followed by amplification using thepolymerase chain reaction (PCR). Insect assays, using tobacco hornworm(Manduca secta) neonate larvae, indicated a significant reduction ofleaf consumption in the transgenic plant when compared to a controlpotato plant and stunted larvae.

Materials and Method

Plasmid

The binary plasmid pWB139 was obtained from Dr. W. M. Barnes ofWashington University, St. Louis, Mo. It contains a translational fusionencoding the 68 kD Bacillus thuringiensis toxin from Bacillusthuringiensis var. kurstaki HD-73 (Adang, M. J., et al., Gene, 36,289-300 (1985)) and neomycin phosphotransferase II from the E. colitransposon Tn5 as shown in FIG. 1. This fused gene is regulated by thecauliflower mosaic virus 35S promoter and the 3' region of the tomatoproteinase inhibitor I (Tom PI 3') gene. This cassette is furtherflanked by the nopaline synthase (NOS) promoter and terminator. Thisplasmid is based on the broad host-range vector pRK252 (Ditta, G., etal., Plasmid, 13, 149-153 (1985)) and is identical to pWB146 (Barnes, W.M., Proc. Natl. Acad. Sci. USA, 87, 9183-9187 (1990)), except that theBacillus thuringiensis var. kurstaki endotoxin codons 1 through 612replace the firefly luciferase codons of pWB146. In pWB139, only codons1-612 from the full length btkgene are used. The reason for not usingthe full length btk codons is that the amino terminal 68 kD of thepeptide has previously been shown to be sufficient for biologicalactivity (Adang, M. J., et al., Gene 36, 289-300 (1985)). Thenucleotides encoding the bt protein in pWB139 are shown in FIGS. 1A-1 to1A-3 in the region between 387 to 2233 as disclosed above.

Transformation, Regeneration, and Selection

Leaf sections (15 mm in width) from potato selection FL1607 (obtained asaxenically-grown plantlets) were inoculated with a ten-fold dilution ofan overnight culture of Agrobacterium tumefaciens strain Wag11containing pWB139, following procedures previously described (Horsch, R.B., et al., Plant Molecular Biology Manual, Kluwer Academic Publishers,Dordrecht, pp. A5:1-9 (1989)). This potato plant has a hightransformation rate with Agrobacterium tumefaciens and thus is easier touse than other plants. The media used for shoot regeneration were fromWenzler et al (Wenzler, H., et al., Plant Science, 63, 79-85 (1989)),except that stage II medium contained 250 mg/l augmentin (BeechamLaboratories, Bristol, Tenn.). Regenerated shoots were transferred toMurashige and Skoog (MS) medium (Murashige, T., et al., Physiol. Plant.,15, 473-497 (1962)) supplemented with 50 mg/l kanamycin for selection.Cultures were maintained in a tissue culture room at 22±2° C. under alight intensity of 60μE m⁻² sec⁻¹ and a photoperiod of 16 hr/dayprovided by cool white fluorescent light.

Screening of Putative Transformants Using PCR

The PCR was performed using a DNA Thermal Cycler (Perkin Elmer Cetus)for 30 cycles of 2 minutes at 95° C., 2 minutes at 65° C., and 5 minutesat 72° C. The PCR reaction included 20 mM Tris buffer (pH 8.55), 2.5 mMMgCl₂, 16 mM (NH₄)₂ SO₄, and 0.15 mg/ml bovine serum albumin (BSA), 0.2mM dNPT's, 5 units Taq polymerase (Biotechnology Systems, New EnglandResearch Products, Boston, Mass.), 20 pM of each of the primers (WB241and WB242), and 0.25 μg sample DNA in a reaction volume of 100 μl. Theprimers WB241 and WB242 have 100% homology to their target templates andamplify a 1090 base pair region of the btk:npt construct. The primersWB241 and WB242 are 30 and 32 base pairs in length, respectively. Theirnucleotide sequences are 5'-TGATCTGGACGAAGAGCATCAGGGGCTCGC (WB241) and5'-TATTGCCAAATGTTTGAAC (WB242).

The amplified DNA was subjected to electrophoresis in a 1% (w/v) agarosegel at 5 v/cm for 1.5 hours and blotted into a Nytran membrane usingcapillary transfer in 10× sodium chloride 0.18 M; sodium phosphate;0.01M ethylene diamine tetramine (EDTA) 0.001M (SSPE) (Sambrook, J., etal., Molecular Cloning: A Laboratory Manual, second edition, Cold SpringHarbor Laboratory Press, Cold Spring Harbor (1988). Hybridization wasperformed following a modification of Southern's procedure (Southern, E.M., J. Mol. Biol. 98, 503-517 (1975)) provided by Schleicher & Schuell,Inc. (Publication No. 77). In this procedure, the membrane containingimmobilized DNA was treated for two hours in a pre-hybridizationsolution containing 6× SSPE, 0.5% (w/v) sodium dodecyl sulfate (SDS),0.2% (w /v) BSA, and 0.2% (w/v) ficol (MW 400,000). The membrane wasthen hybridized to a ³² P-labeled 2.6 kb Hind III fragment of pWB139which encodes the entire translational fusion. This fragment was labeledby random primed labeling (Feinberg, A. P., et al., Annu. Biochem., 132,6-13 (1883)) using a kit from DuPont (Wilmington, Del.). Hybridizationwas performed inside a plastic bag containing a 25 ml solution of 6×SSPE, 0.5% (w/v) SDS, and approximately 5-20 ng/ml of the probe DNA andwas carried out at 60° C. overnight. After hybridization, the membranewas washed twice in 6× SSPE and 0.5% (w/v) SDS at room temperature for15 minutes, then twice in 1× SSPE and 0.5% (w/v) SDS at 37° C. for 15minutes, and finally in 0.1× SSPE and 0.5% (w/v) SDS at 65° C. for 30minutes. Autoradiography was performed using Kodak X-omat AR-5 filmexposed at -70° C. for 2 hours with an intensifying screen (LightningPlus; DuPont, Wilmington, Del.), and developed in an automatic filmdeveloper.

Confirmation of Gene Integration by Southern Blot Hybridization ofRestricted DNA

Total DNA was isolated from transformed and untransformed FL1607 potatoplants as described (Rogers, S. O., et al., Extraction of DNA from planttissues, in: S. B. Gelvin and R. A. Schilperoort (Eds), Plant MolecularBiology Manual, Kluwer Academic Publishers, Dordrecht, pp. A6: 1-11(1989)). DNA (250 μg) from each plant was digested with 150 units of therestriction enzymes Bam HI or Hind III at 37° C. overnight. The digestedDNA was subjected to electrophoresis in a 0.8% (w/v) agarose gel at 1.5v/cm overnight. The procedures for Southern blot hybridization andautoradiography were the same as described earlier in the PCR analysis,except that the film was exposed for seven days at -70° C. Forreconstruction studies pWB139 plasmid DNA (250, 1250, and 2500 pg,corresponding to 1, 5, and 10 copies of the translational fusion in 250μg potato DNA) was digested with Bam HI and run parallel to the potatoDNA.

Detection of the NPT II mRNA in the Transgenic Potato Plant

Fully expanded young leaves (10 g) were collected from transgenic andcontrol potato and tobacco (provided by Dr. Wayne Barnes, WashingtonUniversity, St. Louis, Mo. and used as positive control in mRNApurification and cDNA synthesis) plants. Because expression of thetranslational fusion in tobacco is partially regulated by the 3'terminator of tomato proteinase inhibitor I and is wound-enhanceable,the leaves were pricked with a scalpel and kept overnight in a covered250 ml glass beaker lined with two pieces of moist filter paper. Thefollowing morning, the leaves were ground in liquid nitrogen and totalRNA was extracted according to Gilmour et al (Gilmour, S. J., et al.,Plant Physiol., 87, 745-750 (1988)). Poly A+ mRNA was purified usingHybond-mAP affinity paper (Wreschner, D. H., et al., Nucl. Acids Res.12, 1349-1359 (1984)). First strand cDNA was synthesized in a 50 μlreaction buffer containing 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mMMgCl₂, 10 mM dithiothreitol, 50 μg/ml oligo (dT)₁₂₋₁₈, 500 μM each dATP,dGTP, dCTP, and dTTP, 2.5 μl (500 units) M-MLV reverse transcriptase(Bethesda Research Laboratory, Life Technologies, Inc.), and 5 μgpurified poly A⁺ mRNA sample. The reactions were incubated at 37° C. forone hour, then terminated by adding 1 μl of 0.25 M Na₂ EDTA (pH 7.5) andplaced on ice. A 5 μl sample of the reaction was used as template in thesubsequent PCR amplification. The conditions for the PCR were the sameas described earlier, except that the pair of primers used (MB23 andMB24) amplify a different region (720 bp) of the btk:npt fusion. Theprimers MB23 and MB24 and 29 and 25 base pairs in length respectively.Their nucleotide sequences are 5'-GCTATGACTGGGCACAACAGACAATCGGC for MB23and 5'-CTCGTCAAGAAGGCGATAGAAGGCG for MB24. Electrophoresis, Southernblot hybridization, and autoradiography were conducted using the sameprocedures described earlier for PCR analysis. To check DNAcontamination in the mRNA preparations, parallel cDNA synthesis reactionand PCR were performed without reverse transcriptase.

Insect Assays

Bioassays were conducted on leaf disks (2 cm diameter) punched out fromfully expanded young leaves of transformed and control potato plants inthe morning in the greenhouse. The leaf disks were placed singly inmoist, filter paper-lined, polystyrene petri dishes (50 ×9 mm, withsnap-on lids) and brought to the laboratory. The initial area of eachdisk was measured on a portable leaf area meter (Model L1-3000, LambdaInstruments Corporation, USA). The following day, each leaf disk wasinfested with five healthy, neonate tobacco hornworm larvae. After 24hours, larvae were removed from petri dishes and the area of each leafdisk was measured on the area meter. The difference between the initialand the final leaf areas represented the leaf area consumed and was usedas an indicator of insect feeding. Since variations in thickness ofpotato leaves could mask feeding differences, the dry weight of insectfrass was also used as a parameter of insect feeding. Insect frasscollected from each petri dish was dried for 72 hours and weighed on amicrobalance. Larval mortality, if any, was also recorded.

Insect feeding activity was measured on six leaf disks from thetransformed potato plant and 33 leaf disks from the control potatoplant. All feeding tests were conducted at 25° to 27° C., 65 to 75%relative humidity, and under a 12:12 hour (light:dark) photoperiod.Statistical differences between feeding activity on transformed andcontrol leaf disks were computed by the General Linear Model procedureof Analysis of Variance and the means were compared by Tukey's test(Wilkinson, L., SYSTAT: The system for statistics, SYSTAT, Inc., Prints,Evanston, Ill., pp. 677 (1990)).

Results and discussion

In vitro shoot regeneration from leaf explants inoculated withAgrobacterium was efficient. Callus formed on the edges of leaf explantscultured on stage I medium two to three weeks following bacterialinoculation. After another four to six weeks on stage II medium, shootswere regenerated from the callus tissue. Two hundred and forty-threeshoots were regenerated from fifty leaf explants. Twenty-four of theseshoots were able to produce roots (3 to 9 mm in length) in the mediumcontaining 50 mg/l kanamycin, while none of the 25 shoots fromuntransformed control plants produced roots when cultured in the sameselection medium.

The presence of the btk:npt gene fusion was detected by Southern blothybridization of PCR amplified DNA from leaf samples in three of theseven selected plants (FIG. 2). Screening for transgenic plants by PCRis efficient, although a positive signal detected by PCR does notnecessarily mean that the gene has been integrated into the plantgenome. Based on the results of PCR analysis, three plants which werepositive by PCR were further analyzed for integration of the gene intothe plant genome. Southern blot hybridization of Hind III digested totalDNA from one of these plants, revealed a 2.6 kb band (FIG. 3),confirming the results of PCR that the btk:neo fusion was present in theplant genome. There were two bands (5 and 14 kb each) demonstrated inthe Bam HI digestion. Because Bam HI recognizes a unique site in theplasmid pWB139, this result suggests at least two independentintegration events. Results of the reconstruction analysis indicate thatfive to ten copies of the gene fusion are integrated, possibly in atandem fashion. Therefore, it is concluded that the btk:npttranslational fusion has been integrated into the genome of this plantwithout any major structural alterations within the translationalfusion. However, two of the three plants which were positive by PCR didnot display any positive signal by genomic Southern analysis (data notshown). The discrepancy between PCR and Southern blot of the restrictedgenomic DNA may be due to a very low proportion of cells beingtransformed.

Expression of the translational fusion in the transgenic potato plantwas detected by Southern blot hybridization of PCR amplified cDNAsynthesized from purified mRNA of the transgenic potato plant (FIG. 4).The possibility of DNA contamination was ruled out by the fact that noDNA was detected after PCR amplification without adding reversetranscriptase to the reaction.

Insect feeding differed distinctly between leaf disks of transformed andcontrol plants as shown in FIG. 5. The neonate tobacco hornworm larvaeconsumed significantly more leaf area on leaf disks from the controlpotato plant than on leaf disks taken from the transformed potato (TableI). Also, frass collected from petri dishes containing transgenic potatoleaf disks weighed significantly less than that collected from dishescontaining the control leaves (Table I). This difference also indicatesreduced feeding on the transformed potato plant. Although few neonatesdied after 24 hour confinement in the petri dishes, those that fed onleaf disks of the transformed potato plant were smaller, sluggish, andpale, compared with those that fed on the control leaf disks (FIG. 5).FIG. 6 shows the relative size of the hookworms for a control (upper)and transgenic plant feedings (two lower). Thus, transformed potatoplant foliage was relatively more resistant to tobacco hornworm feeding.

                  TABLE I                                                         ______________________________________                                        Mean leaf area consumed by five neonate                                         tobacco hornworm larvae in 24 hours and mean dry weight                       of frass on leaf disks of transformed and untransformed                       FL1607 plants..sup.a,b                                                        Plant       Leaf area (cm.sup.2 ± SD)                                                                 Frass weight (μg ± SD)                     ______________________________________                                          Transformed 0.61 ± 0.205 0.82 ± 0.404                                   Untransformed 1.86 ± 0.373 2.89 ± 0.769                               ______________________________________                                         .sup.a Means of six replications of transformed and 33 replications of        control potato leaf disks;                                                    .sup.b Means of transformed and untransformed in a column are                 significantly different (p < 0.001) using Tukey's procedure.             

Expression of full-length bt genes in transgenic plants has beenreported to be extremely low (Hofte, H. and H. R. Whiteley,Microbiological Reviews, 53, 242-255 (1989); and Vaeck, M., et al.,Nature, 328, 33-37 (1987)). We also found low bt-expression in thetransgenic potato plant and were unable to detect bt mRNA with Northernblot hybridization, although the bt gene used is truncated and abiologically active portion of the full length toxin (Adang, M. J., etal., Gene, 36, 289-300 (1985)). However, using PCR amplification of cDNAfrom reverse transcribed mRNA, we detected the expression of the btk:neotranslational fusion in the transgenic potato plant.

It has been reported that degrees of insect resistance of transgenicplants are positively correlated with bt mRNA levels in the transgenicplants (Vaeck, M., et al., Nature, 328, 33-37 (1987)) and thattransgenic tobacco and tomato plants with low btk gene expression stillhave substantial tolerance to the tobacco hornworm and other targetinsects (Vaeck, M., et al., Nature, 328, 33-37 (1987); and Delannay, X.,et al., Bio/Technology, 7, 1265-1269 (1989)). In the present case,higher expression produces better results. The insect resistance of thetransgenic potato plant demonstrated in the potato plants isparticularly useful in reducing the amount of synthetic pesticides usedin protecting potato crops worldwide.

The transgenic potato plant and seed potatoes are on deposit fromMichigan State University, East Lansing, Mich. as a depository.

It is intended that the foregoing description be only illustrative ofthe present invention and that the present invention be limited only bythe hereinafter appended claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 1                                           - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  1836                                                             (B) TYPE:  Nucleic A - #cid                                                   (C) STRANDEDNESS:  Sing - #le                                                 (D) TOPOLOGY:  Linear                                                - -     (ii) MOLECULE TYPE: Plasmid DNA                                       - -    (iii) HYPOTHETICAL: No                                                 - -     (iv) ANTI-SENSE: No                                                   - -      (v) FRAGMENT TYPE:  N-terminal, inter - #nal and C-terminal                       fragments                                                       - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM:  Bacillus - # thuringiensis                                     (B) STRAIN:  N/A                                                              (C) INDIVIDUAL ISOLATE: - # pWB139                                            (D) DEVELOPMENTAL STAGE: - #N/A                                               (E) HAPLOTYPE:  N/A                                                           (F) TISSUE TYPE: N/A                                                          (G) CELL TYPE: N/A                                                            (H) CELL LINE: N/A                                                            (I) ORGANELLE:  N/A                                                  - -    (vii) IMMEDIATE SOURCE:  N/A                                           - -   (viii) POSITION IN GENOME:  N/A                                         - -     (ix) FEATURE:                                                                  (A) NAME/KEY: endotoxin - #encoding DNA                                       (B) LOCATION: BAM III - #to XhoI                                                   DNA fragm - #ent in pB139                                                (C) IDENTIFICATION METHOD: - # sequencing                                     (D) OTHER INFORMATION: - #DNA needed for endotoxin                                 expression.                                                     - -      (x) PUBLICATION INFORMATION: Barnes, W. - #M., Proc. Natl.         Acad. Sci.                                                                                     US, 87, - #9183-9187 (1990)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - -                   - #                  - #                  - #        ATGGATCCCA     10                                                                                 - #                  - #                  - #             MetAsnProL                                                                       - - AGCTTAACAT CAATGAATGC ATTCCTTATA ATTGTTTAAG TAACCCTGAA GT -            #AGAAGTAT     70                                                                ysLeuAsnIl eAsnGluCys IleProTyrA snCysLeuSe rAsnProGlu Va - #lGluValL          5            10    - #          15        - #     20             - #25       - - TAGGTGGAGA AAGAATAGAA ACTGGTTACA CCCCAATCGA TATTTCCTTG TC -             #GCTAACGC    130                                                                euGlyGlyGl uArgIleGlu ThrGlyTyrT hrProIleAs pIleSerLeu Se - #rLeuThrG                    30      - #        35          - #     40             - # 45       - - AATTTCTTTT GAGTGAATTT GTTCCCGGTG CTGGATTTGT GTTAGGACTA GT -             #TGATATAA    190                                                                lnPheLeuLe uSerGluPhe ValProGlyA laGlyPheVa lLeuGlyLeu Va - #lAspIleI                    50      - #        55          - #     60             - # 65       - - TATGGGGAAT TTTTGGTCCC TCTCAATGGG ACGCATTTCT TGTACAAATT GA -             #ACAGTTAA    250                                                                leTrpGlyIl ePheGlyPro SerGlnTrpA spAlaPheLe uValGlnIle Gl - #uGlnLeuI                    70      - #        75          - #     80             - # 85       - - TTAACCAAAG AATAGAAGAA TTCGCTAGGA ACCAAGCCAT TTCTAGATTA GA -             #AGGACTAA    310                                                                leAsnGlnAr gIleGluGlu PheAlaArgA snGlnAlaIl eSerArgLeu Gl - #uGlyLeuS                    90      - #        95          - #     100            - #        105                                                                              - - GCAATCTTTA TCAAATTTAC GCAGAATCTT TTAGAGAGTG GGAAGCAGAT CC -            #TACTAATC    370                                                                erAsnLeuTy rGlnIleTyr AlaGluSerP heArgGluTr pGluAlaAsp Pr - #oThrAsnP                    110      - #       115          - #    120             -         #125                                                                             - - CAGCATTAAG AGAAGAGATG CGTATTCAAT TCAATGACAT GAACAGTGCC CT -            #TACAACCG    430                                                                roAlaLeuAr gGluGluMet ArgIleGlnP heAsnAspMe tAsnSerAla Le - #uThrThrA                    130      - #       135          - #    140             -         #145                                                                             - - CTATTCCTCT TTTTGCAGTT CAAAATTATC AAGTTCCTCT TTTATCAGTA TA -            #TGTTCAAG    490                                                                laIleProLe uPheAlaVal GlnAsnTyrG lnValProLe uLeuSerVal Ty - #rValGlnA                    150      - #       155          - #    160             -         #165                                                                             - - CTGCAAATTT ACATTTATCA GTTTTGAGAG ATGTTTCAGT GTTTGGACAA AG -            #GTGGGGAT    550                                                                laAlaAsnLe uHisLeuSer ValLeuArgA spValSerVa lPheGlyGln Ar - #gTrpGlyP                    170      - #       175          - #    180             -         #185                                                                             - - TTGATGCCGC GACTATCAAT AGTCGTTATA ATGATTTAAC TAGGCTTATT GG -            #CAACTATA    610                                                                heAspAlaAl aThrIleAsn SerArgTyrA snAspLeuTh rArgLeuIle Gl - #yAsnTyrT                    190      - #       195          - #    200             -         #205                                                                             - - CAGATTATGC TGTACGCTGG TACAATACGG GATTAGAACG TGTATGGGGA CC -            #GGATTCTA    670                                                                hrAspTyrAl aValArgTrp TyrAsnThrG lyLeuGluAr gValTrpGly Pr - #oAspSerA                    210      - #       215          - #    220             -         #225                                                                             - - GAGATTGGGT AAGGTATAAT CAATTTAGAA GAGAATTAAC ACTAACTGTA TT -            #AGATATCG    730                                                                rgAspTrpVa lArgTyrAsn GlnPheArgA rgGluLeuTh rLeuThrVal Le - #uAspIleV                    230      - #       235          - #    240             -         #245                                                                             - - TTGCTCTGTT CCCGAATTAT GATAGTAGAA GATATCCAAT TCGAACAGTT TC -            #CCAATTAA    790                                                                alAlaLeuPh eProAsnTyr AspSerArgA rgTyrProIl eArgThrVal Se - #rGlnLeuT                    250      - #       255          - #    260             -         #265                                                                             - - CAAGAGAAAT TTATACAAAC CCAGTATTAG AAAATTTTGA TGGTAGTTTT CG -            #AGGCTCGG    850                                                                hrArgGluIl eTyrThrAsn ProValLeuG luAsnPheAs pGlySerPhe Ar - #gGlySerA                    270      - #       275          - #    280             -         #285                                                                             - - CTCAGGGCAT AGAAAGAAGT ATTAGGAGTC CACATTTGAT GGATATACTT AA -            #CAGTATAA    910                                                                laGlnGlyIl eGluArgSer IleArgSerP roHisLeuMe tAspIleLeu As - #nSerIleT                    290      - #       295          - #    300             -         #305                                                                             - - CCATCTATAC GGATGCTCAT AGGGGTTATT ATTATTGGTC AGGGCATCAA AT -            #AATGGCTT    970                                                                hrIleTyrTh rAspAlaHis ArgGlyTyrT yrTyrTrpSe rGlyHisGln Il - #eMetAlaS                    310      - #       315          - #    320             -         #325                                                                             - - CTCCTGTAGG GTTTTCGGGG CCAGAATTCA CTTTTCCGCT ATATGGAACT AT -            #GGGAAATG   1030                                                                erProValGl yPheSerGly ProGluPheT hrPheProLe uTyrGlyThr Me - #tGlyAsnA                    330      - #       335          - #    340             -         #345                                                                             - - CAGCTCCACA ACAACGTATT GTTGCTCAAC TAGGTCAGGG CGTGTATAGA AC -            #ATTATCGT   1090                                                                laAlaProGl nGlnArgIle ValAlaGlnL euGlyGlnGl yValTyrArg Th - #rLeuSerS                    350      - #       355          - #    360             -         #365                                                                             - - CCACTTTATA TAGAAGACCT TTTAATATAG GGATAAATAA TCAACAACTA TC -            #TGTTCTTG   1150                                                                erThrLeuTy rArgArgPro PheAsnIleG lyIleAsnAs nGlnGlnLeu Se - #rValLeuA                    370      - #       375          - #    380             -         #385                                                                             - - ACGGGACAGA ATTTGCTTAT GGAACCTCCT CAAATTTGCC ATCCGCTGTA TA -            #CAGAAAAA   1210                                                                spGlyThrGl uPheAlaThr GlyThrSerS erAsnLeuPr oSerAlaVal Ty - #rArgLysS                    390      - #       395          - #    400             -         #405                                                                             - - GCGGAACGGT AGATTCGCTG GATGAAATAC CGCCACAGAA TAACAACGTG CC -            #ACCTAGGC   1270                                                                erGlyThrVa lAspSerLeu AspGluIleP roProGlnAs nAsnAsnVal Pr - #oProArgG                    410      - #       415          - #    420             -         #425                                                                             - - AAGGATTTAG TCATCGATTA AGCCATGTTT CAATGTTTCG TTCAGGCTTT AG -            #TAATAGTA   1330                                                                lnGlyPheSe rHisArgLeu SerHisValS erMetPheAr gSerGlyPhe Se - #rAsnSerS                       430   - #          435       - #       440                      - - GTGTAAGTAT AATAAGAGCT CCTATGTTCT CTTGGATACA TCGTAGTGCT GA -             #ATTTAATA   1390                                                                erValSerIl eIleArgAla ProMetPheS erTrpIleHi sArgSerAla Gl - #uPheAsnA        445            450     - #        455         - #     460                      - - ATATAATTGC ATCGGATAGT ATTACTCAAA TCCCTGCAGT GAAGGGAAAC TT -             #TCTTTTTA   1450                                                                snIleIleAl aSerAspSer IleThrGlnI leProAlaVa lLysGlyAsn Ph - #eLeuPheA        465            470     - #        475         - #     480                      - - ATGGTTCTGT AATTTCAGGA CCAGGATTTA CTGGTGGGGA CTTAGTTAGA TT -             #AAATAGTA   1510                                                                snGlySerVa lIleSerGly ProGlyPheT hrGlyGlyAs pLeuValArg Le - #uAsnSerS        485            490     - #        495         - #  500             505         - - GTGGAAATAA CATTCAGAAT AGAGGGTATA TTGAAGTTCC AATTCACTTC CC -             #ATCGACAT   1570                                                                erGlyAsnAs nIleGlnAsn ArgGlyTyrI leGluValPr oIleHisPhe Pr - #oSerThrS                    510      - #       515          - #    520             -         #525                                                                             - - CTACCAGATA TCGAGTTCGT GTACGGTATG CTTCTGTAAC CCCGATTCAC CT -            #CAACGTTA   1630                                                                erThrArgTy rArgValArg ValArgTyrA laSerValTh rProIleHis Le - #uAsnValA                    530      - #       535          - #    540             -         #545                                                                             - - ATTGGGGTAA TTCATCCATT TTTTCCAATA CAGTACCAGC TACAGCTACG TC -            #ATTAGATA   1690                                                                snTrpGlyAs nSerSerIle PheSerAsnT hrValProAl aThrAlaThr Se - #rLeuAspA                    550      - #       555          - #    560             -         #565                                                                             - - ATCTACAATC AAGTGATTTT GGTTATTTTG AAAGTGCCAA TGCTTTTACA TC -            #TTCATTAG   1750                                                                snLeuGlnSe rSerAspPhe GlyTyrPheG luSerAlaAs nAlaPheThr Se - #rSerLeuG                    570      - #       575          - #    580             -         #585                                                                             - - GTAATATAGT AGGTGTTAGA AATTTTAGTG GGACTGCAGG AGTGATAATA GA -            #CAGATTTG   1810                                                                lyAsnIleVa lGlyValArg AsnPheSerG lyThrAlaGl yValIleIle As - #pArgPheG                    590      - #       595          - #    600             -         #605                                                                             - - AATTTATTCC AGTTACTGCA ACACTC          - #                  - #                1836                                                                    luPheIlePr oValThrAla ThrLeu                                                              610                                                              __________________________________________________________________________

We claim:
 1. A transformed potato plant comprising DNA as set forth inSEQ ID NO:1 which encodes a Bacillus thuringiensis endotoxin, whereinfive to ten copies of the DNA are integrated into the genome of theplant and wherein the endotoxin is expressed at a level such that theplant is resistant to Lepidopteran insects.
 2. The transformed potatoplant of claim 1 wherein the potato plant prior to transformation isSolanum tuberosum FL
 1607. 3. The transformed potato plant of claim 1wherein the DNA has been transformed into the plant using Agrobacteriumtumefaciens as a vector.
 4. The transformed potato plant of claim 1wherein the DNA is linked to a marker DNA which allows selection for thetransformed plant.
 5. The transformed potato plant of claim 4 whereinthe marker DNA encodes neomycin phosphotransferase II.
 6. Thetransformed potato plant of claim 1 wherein the potato plant prior totransformation is Solanum tuberosum FL1607, wherein the DNA has beentransformed into the plant using Agrobacterium tumefaciens as a vector,and wherein the DNA is linked to a marker DNA which allows selection forthe transformed plant.
 7. The transformed potato plant of claim 6wherein the marker DNA encodes neomycin phosphotransferase II.
 8. Thetransformed potato plant of claim 1 wherein the DNA is operably linkedto a polyadenylation encoding DNA region.
 9. The transformed potatoplant of claim 8 wherein the polyadenylation encoding DNA regioncomprises Tom PI 3' and NOS 3'.
 10. A method for producing a transformedpotato plant which is resistant to Lepidopteran insects comprising:(a)transforming potato plants with DNA encoding a Bacillus thuringiensisendotoxin as set forth in SEQ ID NO:1; and (b) selecting a transformedpotato plant, wherein five to ten copies of the DNA are integrated intothe genome of the plant and wherein the endotoxin is expressed at alevel such that the plant is resistant to Lepidopteran insects.
 11. Themethod of claim 10 wherein the transformed potato plant prior totransformation is Solanum tuberosum FL
 1607. 12. The method of claim 10wherein the DNA is transformed into the plant using Agrobacteriumtumefaciens as a vector.
 13. The method of claim 10 wherein the DNA islinked to a marker DNA which allows selection for the transformed plant.14. The method of claim 13 wherein the marker DNA encodes neomycinphosphotransferase II.
 15. The method of claim 10 wherein thetransformed potato plant prior to transformation is Solanum tuberosumFL1607, wherein the DNA is transformed into the plant usingAgrobacterium tumefaciens as a vector, and wherein the DNA is linked toa marker DNA which allows selection for the transformed plant.
 16. Themethod of claim 15 wherein the marker DNA encodes neomycinphosphotransferase II.
 17. The method of claim 10 wherein the DNA isoperably linked to a polyadenylation encoding DNA region.
 18. The methodof claim 17 wherein the polyadenylation encoding DNA region comprisesTom PI 3' and NOS 3'.